Protein interaction networks are key determinants of protein function in biological systems. Despite the potential that quantitative protein interaction information could have for all areas of cancer research, global or large-scale quantitation of protein interactions within native living systems is a challenge that is unmet by today's technology. Improved capabilities to quantitate protein interactions will have a major impact on the understanding of cancer, metastasis and the development of anti-cancer drug resistance, among many other areas. This project aims to develop and apply novel Protein Interaction Reporter (PIR) technology in conjunction with stable isotope labeling and label-free methods to enable large-scale quantitation of protein interactions and topological features in cancer cells. PIRs are chemical cross-linkers that possess cleavable bonds that allow the compounds to be dissociated with high specificity within the mass spectrometer. This allows complex cross-linking products that result from on-cell cross-linking reactions to be measured as an intact complex and then, identified through the detection of an expected mass reporter ion and intact peptide ions that are released upon PIR activation. In this project, PIR compounds will be synthesized and used to identify protein interactions in cancer cells using mass spectrometry. This will be done by establishing cross-linked peptide connectivity through expected PIR mass relationships and targeted MS/MS on released peptides to identify proteins. The next phase of research will involve the combined application of PIR technology with isotope labeling and label-free methods to cancer cells, cells that have been treated with anticancer drugs, and cancer cells that have developed drug resistance. Successful application of this technology will for the first time, demonstrate the potential for large-scale quantitation of protein interactions in cells and will identify protein interactions relevant to cancer cell drug resistance.